Copper status and its relation to abdominal obesity indices and liver function in non-alcoholic fatty liver disease: a case-control study

Abstract

Objective

This study investigated copper (Cu) status in relation to abdominal obesity indices and liver function in patients with non-alcoholic fatty liver disease (NAFLD). This case-control study was carried out on 80 overweight/obese patients with NAFLD and 80 apparently healthy age, sex, and body mass index (BMI)-matched controls. A validated and reliable 168-item semi-quantitative food frequency questionnaire was completed for each subject and fasting serum levels of liver aminotransferases, ferritin, Cu and ceruloplasmin were assessed.

Results

Mean intakes of energy and carbophydrate were significantly lower in patients with NAFLD than the control group while mean protein intake was highre (p < 0.05). Although mean Cu intake was greater in cases than controls, low dietary intake of Cu was found in 7.5% and 32.5% of the cases and controls, respectively. Apart from serum levels of liver aminotransferases (p < 0.001) and ferritin (p = 0.010), no significant differences were found in serum levels of Cu and ceruloplasmin. Serum and dietary Cu were positively correlated with obesity indices and serum ceruloplasmin was correlated with waist to height ratio and ferritin only in cases (p < 0.05). Low Cu intake (< 0.95 mg/day) was more likely to increase the odds of NAFLD (p for trend = 0.002), after adjusting for potential confounders.

Introduction

Non-alcoholic fatty liver disease (NAFLD)- as one of the most common chronic liver diseases and the leading cause of cirrhosis and hepatocellular carcinoma worldwide- is characterized by excessive fat accumulation in hepatocytes in absence of alcohol and drug abuse as well as steatogenic medications, or other conditions [1]. Around one billion people worldwide are influenced by NAFLD and its prevalence in Iran in general population was estimated 38.07% in 2019 [2, 3]. NAFLD is a multi-factorial condition resulting from genetic, metabolic, environmental, and nutritional factors [1]. NAFLD is closely associated with chronic clinical features such as dyslipidemia, insulin resistance (IR), metabolic syndrome and type 2 diabetes (T2DM) [4]. Nowadays, the pathogenesis of NAFLD is known as a consequence of a “multi-hit process” which starts with free fatty acids (FFAs) metabolism and IR (initial hit) led to non-alcoholic steatohepatitis (NASH), followed by oxidative stress and inflammation in the liver cells (second hit), necrosis accompanied by the activation of fibrogenic cascade which result in fibrosis and even cirrhosis [5]. Furthermore, factors e.g. endotoxinemia, Kupffer cell activation, adipocytokines, mitochondrial dysfunction, oxidative stress and lipid peroxidation by stimulating inflammation lead to progressive liver disease including cell death, liver fibrosis and cirrhosis [5, 6]. There is evidence that a number of essential micronutrients play an important role in ameliorating reactive oxygen species (ROS) generation [7].

Copper (Cu)- as an essential mineral and cofactor- has a pivotal role in many physiological redox reactions, not only in exerting many effects on the liver through cellular respiratory systems but also as a biometal in form of coenzyme in oxidation-reduction processes, iron mobilization and lipid metabolism [7–9]. Cu is a component in the structure of super oxide dismutase (SOD) to detoxicate ROS in neurotransmitter synthesis [10]. Moreover, Cu inadequacy appears to be associated with obesity and its related conditions such as metabolic syndrome [11]. There is animal and human evidence demonstrating the link between low serum Cu concentration (less than 70 µg/dl) and fat accumulation in the liver [11–13]. Cu deficiency or hypocupremia- defined as insufficient Cu to meet the needs of the body or as low serum Cu level or ceruloplasmin – is related to symptoms such as myelodysplasia, anemia, neurological problems known as “swayback” in ruminant animals, myopathies, ataxia, and Menkes disease [14, 15]. Although World Health Organization (WHO) has suggested that the minimal acceptable intake of Cu is 0.9–1.3 mg/day, the average consumption is estimated ~ 2 mg/day [16]. Food sources of Cu are varied, however, only 20–50% of dietary intake (~ 5 mg/day) is absorbed [14].

There are a number of studies investigating the relationship between Cu hemostasis dysfunction and some chronic diseases [9, 13, 17]. In the study by Aigner et al. [9], reduced hepatic Cu concentrations were observed in those with NAFLD and were associated with more pronounced hepatic steatosis, NASH, and components of the metabolic syndrome. Indeed, Nobili et al. [17] by assessing Cu, iron, ceruloplasmin concentration and activity, transferrin (Tf), ferroxidase activity, and ferritin, as well as transferrin saturation and ceruloplasmin to Tf ratio as an index of the activity of the antioxidant ceruloplasmin -Tf system in 100 children with NAFLD confirmed by biopsy showed that ceruloplasmin failure-related antioxidant capacity is strongly associated with NAFLD-related damage. Nunes et al. [13] in their study on 95 patients with NAFLD confirmed by biopsy also reported that body mass index (BMI) and low-density lipoprotein cholesterol (LDL-C) were lower while total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and ferritin concentrations were higher in patients with low Cu status compared with those with adequate Cu. Moreover, oxidative stress and the pro-oxidant-antioxidant balance of the body as well as increased lipid synthesis could be influenced by Cu deficiency in NAFLD progression [8, 9, 12, 17]. In 2018, Song et al. [18] implied the role of Cu homeostasis in relation to high fructose intake in terms of Cu-fructose interactions in fructose-induced NAFLD. However, in heopatocellular carcinoma, Porcu et al. [19] showed higher serum Cu concentrations in presence of liver cancer. Changes in Cu hemostasis have been shown to be involved in some chronic metabolic diseases such as NAFLD, strictly in relation to its effect on oxidative stress [20].

As the prevalence of NAFLD is increasing rapidly worldwide and the results of studies examining the association between Cu deficiency and NAFLD are inconsistent, the present study aimed to investigate Cu status in relation to abdominal obesity indices and liver function in NAFLD.

Materials and methods

Study design

The present case-control study was carried out on 160 male and female adults aged 20–60 years with BMI ≥ 25 kg/m² in Tabriz, Iran between October 2019 and May 2020. Eighty patients with mild or moderate NAFLD confirmed by ultrasonography findings [21] as “Cases” and 80 age, sex and BMI-matched apparently healthy subjects as “Controls”. The subjects who were pregnant, lactation, menopause, athlete, bariatric surgery, suffering from other chronic liver diseases, diabetes mellitus, hypertension, autoimmune disorder, renal or cardiovascular diseases, malignancies, taking lipid- or glucose- lowering medications, oral contraceptives, vitamin-mineral supplements over last three months were excluded. The study protocol was approved by the Ethics Committee of Tabriz University of Medical Science (IR.TBZMED.REC.1399.176) and all participants signed an informed written consent form.

Sample size

Aigner et al. [9] study and reported difference in serum Cu level was used for sample size calculation according to the following formula. By considering 95% confidence level and 80% power, 80 subjects in each group was estimated.

N= (Z_1-α/2+ Z_1-β)²( S₁²+ S₂²) / ( µ_{1 -}µ₂)²

Data collection

All subjects were interviewed face-to-face for collecting demographic, socioeconomic status, and behavioral details such as smoking, alcohol drinking, medical history, and taking supplements by a trained nutritionist. Weight and height were measured using a calibrated stadiometer (Seca, Germany) wearing light cloth and without shoes to the nearest 0.1 kg and 0.1 cm, respectively. Waist circumference (WC) and hip circumference (HC) were assessed at the midpoint between the lower border of the rib cage and iliac crest and around the widest part of buttocks to the nearest 0.5 cm using a non-stretchable tape, respectively. Then, obesity indices including BMI, WC to HC ratio (WHR), WC to height ratio (WHtR) were calculated.

Laboratory assay

Five ml blood sample was taken after 12–14 h overnight fasting, then centrifuged and collected serum was stored at − 20 °C until the end of the study. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using Pars Azmoon kit and an auto-analyzer (Hitachi 902, Tokyo, Japan) based on International Federation of Clinical Chemistry (IFCC) procedure [22]. Indeed, serum ferritin level was quantified using electrochemiluminescence immunoassay while serum Cu and ceruloplasmin were assessed using commercial kits and colorimetry and nephelometry assay, respectively. Serum concentration of Cu < 70 µg/dl [8] and ceruloplasmin < 20 g/L, were defined as deficiency [23].

Dietary assessment

For dietary assessment, a validated and reliable 168-item semi-quantitative food frequency questionnaire (FFQ) [24] was used and then revised to a 132-item questionnaire after considering eating habits of the participants for overweight and obese adults in Iran [25]. The FFQ also contains food sources of Cu such as organ meats, sea foods, legumes, cereals, and nuts [25]. The subjects’ food consumption based on frequency per day, week, month or year and portion size at each time during the past year were asked by a trained nutritionist. The frequency consumption of foods for Cu intake was recorded and then transformed per week and add up for each food group regarding estimating dietary intake of Cu. Daily intake of energy, macronutrients, dietary fiber and Cu were estimated using the Nutritionist 4 software (First Databank, San Bruno, CA, USA) modified for Iranian foods. Cu intake was finally classified into quartiles. The WHO defined 0.9–1.3 mg as the minimal Cu daily intake [16].

Statistical analysis

Data was analyzed using Statistical Package for Social Analysis (SPSS Inc., ver. 20, Chicago, IL, USA). To check the normality distribution of continuous variables, Kolmogorov-Smirnov test was applied. Chi-square test was used for the relationship between categorical variables. Differences in symmetric continuous variables were done using two-independent sample t- and analysis of variance (ANOVA) tests between two groups and more than two groups, respectively, while for asymmetric ones, Mann-whitney U- and Kruskall-Wallis test were used. Association between continuous variables were checked using Pearson and Spearman correlation coefficient for symmetric and asymmetric data, respectively. Dietary Cu was classified into quartiles to investigate the relation between dietary Cu intake and NAFLD as well as WHO classification for defining Cu adequacy. The relationship between dietary Cu status with the odds of NAFLD, multivariate logistic regression was used to estimate odds ratios (OR) and 95% confidence interval (CI). P < 0.05 was considered as statistically significant.

Results

In this case-control study, 80 patients with NAFLD and 80 age-sex-and BMI-matched apparently healthy subjects were studied. As Table 1 shows those with NAFLD had significantly greater anthropometric measures and obesity indices compared with the healthy individuals. More than half of the patients suffered from mild NAFLD. High BMI was found in 86.3% of cases and 42.5% of controls (p < 0.001). No significant differences were found in marital status and educational level between the groups.

Table 1.

Characteristics of the participants

	Control (N = 80)	NAFLD (N = 80)	p
	SD ± Mean	SD ± Mean
Age (yrs.)	30.90 ± 9.25	30.24 ± 7.42	0.618*
Weight (kg)	79.19 ±13.48	94.34 ± 18.52	0.001*
Height (cm)	162.89 ± 8.36	161.34 ± 13.42	0.382*
WC (cm)	96.01 ± 13.97	111.35 ± 13.41	0.001*
HC (cm)	108.13 ± 13.59	118.30 ± 10.15	0.001*
WHR	0.89 ±0.08	0.94 ± 0.07	0.001*
WHtR	0.59 ± 0.09	0.70 ± 0.12	0.001*
BMI (kg/m2)	29.89 ± 5.05	38.36 ± 23.22	0.002*
	N (%)	N (%)
Gender, female	66 (82.5)	62 (77.5)	0.429**
Marital status, married	37 (46.3)	46 (57.5)	0.154**
Educational			0.050**
Up to high school	2 (2.5)	8 (10.0)
University degree	78 (97.5)	72 (90.0)
BMI status ((kg/m2)			< 0.001**
25-29.99	46 (57.5)	11 (13.8)
30-39.99	34 (42.5)	69 (86.2)
NAFLD severity			-
Grade I (Mild)	-	47 (58.8)
Grade II (Moderate)	-	33 (41.2)

WC, Waist circumference; HC, Height circumference; WHR, Waist-to-hip ratio; WHtR, Waist-to-height ratio; BMI, Body mass index; NAFLD, Non-alcoholic fatty liver disease. *p for Independent sample t-test; **p for χ² test

Table 2 demonstrates dietary intakes as well serum in cases and controls. Mean intakes of energy and carbophydrate were significantly lower (p = 0.018 and p = 0.007, respectively) in patient with NAFLD than control group while mean protein intake was highre (p = 0.009). Furthermore, according to WHO recommendation [16], low dietary intake of Cu (i.e. <0.90 mg/day) was found in 7.5% and 32.5% of the cases and controls, respectively, while 45.0% of the cases and 32.5% of the controls consumed Cu in adequate amount (i.e. 0.9–1.3 mg/day) (p = 0.001) (Data not shown). Moreover, Cu intake was greater in cases than controls in terms of absolute amount or quartiles of intake (p = 0.005 and p = 0.001, respectively). However, apart from serum levels of liver aminotransferases (p < 0.001) and ferritin (p = 0.010), no significant differences were found in serum levels of Cu and ceruloplasmin (Table 2).

Table 2.

Comparison of dietary intakes and biochemical parameters between two groups

	Control (N = 80)	NAFLD (N = 80)	p
Dietary intakes
Energy (kcal)	1844.7 ± 847.6	1611.4 ± 183.5	0.018*
Carbohydrate (g)	278.2 ± 154.4	229.7 ± 33.2	0.007*
Protein (g)	65.8 ± 19.13	8.6 ± 72.1	0.009*
Fat (g)	52.7 ± 42.1	48.6 ± 8.3	0.395*
Copper (mg)	1.33 ± 1.29	1.40 ± 0.56	0.005**
Q1 (0.15–0.94)	30 (34.7)	10 (12.5)	0.001***
Q2 (0.95–1.22)	19 (23.8)	21 (26.3)
Q3 (1.23–1.46)	12 (15.0)	29 (36.2)
Q4 (1.47–10.37)	19 (23.8)	20 (25.0)
Serum
Cu (µg/dl) ceruloplasmin (mg/dl) Ferritin (ng/ml) ALT (IU/L) AST (IU/L)	111.17 ± 37.75 36.15 ± 11.99 47.41 ± 42.58 17.07 ± 5.19 17.93 ± 4.05	116.37 ± 41.43 38.26 ± 8.63 70.01 ± 65.32 26.11 ± 11.66 21.95 ± 8.62	0.408* 0.204* 0.010** < 0.001* < 0.001*

Cu, Copper; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase. *p for Independent sample t-test; ** p for Mann-Whitney U-test; ***p for χ² test

Only in patients with NAFLD, serum and dietary Cu were positively and significantly correlated with obesity indices [BMI (r = 0.215, p = 0.055 and r = 0.230, p = 0.040), WC (r = 0.277, p = 0.013 and r = 0.236, p = 0.035) and WHtR (r = 0.335, p = 0.002 and r = 0.264, p = 0.018), respectively], while there was a significant correlation between serum levels of ceruloplasmin and WHtR (r= -0.292, p = 0.009). Hence, serum ceruloplasmin concentration was negatively associated with serum ferritin level only in cases (r= -0.292, p = 0.009) (Data not shown).

Table 3 reveals that NAFLD odds was significantly associated with dietary Cu intake as well as BMI, WC and serum levels of ALT, AST and ferritin as confounders. In multivariate model and by considering the confounders, low Cu intake, in a dose-response manner, was more likely to be associated with NAFLD odds (OR = 5.82, CI 95%: 1.55, 21.85, p = 0.009) (p for trend = 0.002).

Table 3.

Predictors of NAFLD

Variables	Unadjusted OR (CI 95%)	P*	Adjusted OR (CI 95%)	P**
Cu dietary intake (mg)		0.007		0.002
Q1 (0.15–0.94)	0.32 (0.12, 0.82)	0.018	5.82 (1.55, 21.85)	0.009
Q2 (0.95–1.22)	1.05 (0.42, 2.54)	0.914	2.41 (0.69, 8.48)	0.169
Q3 (1.23–1.46)	2.30 (0.92, 5.76)	0.077	0.52 (0.15, 1.85)	0.314
Q4 (1.47–10.37)	1.00		1.00
ALT (mg/dL)	1.14 (1.08, 1.20)	< 0.001	1.14 (1.05, 1.25)	0.003
AST (mg/dL)	1.13 (1.06, 1.21)	< 0.001	0.98 (0.87, 1.11)	0.720
Ferritin	1.01 (1.00, 1.02)	0.015	1.00 (0.99, 1.01)	0.471
BMI (kg/m2)	1.24 (1.14, 1.34)	< 0.001	1.01 (0.87, 1.16)	0.939
WC (cm)	1.11 (1.07, 1.15)	< 0.001	1.12 (1.04, 1.19)	0.001

OR, Odds ratio; Cu, Copper; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; BMI, Body mass. index; WC, Waist circumference. *p for univariate logistic Regression. ** p for Multivariate logistic regression djusted for Cu intake, ALT, AST, ferritin, BMI and WC

Discussion

Cu- as a biometal in a number of enzymes involved in vital processes such as oxidative phosphorylation, iron transportation and oxidative stress, has been suggested to be linked with some conditions including impaired insulin metabolism and related chronic diseases [26]. The results of the present case-control study showed that low dietary intake of Cu (i.e. <0.90 mg/day) was found in 7.5% and 32.5% of the cases and controls, respectively while 45.0% and 32.5% consumed Cu in adequate amount. This may be related to a number of factors contributing in Cu status such as age, gender, hormone therapy, dietary intake (particularly excessive dietary intake of fructose and zinc) as well as conditions, e.g. Menkes, celiac, and gastric bypass) [12, 27–29]. Cu dietary intake in around 25% of adults in the USA and Canada is lower than the estimated average requirement (0.7 mg/day) [30, 31]. Indeed, low Cu intake has been reported in Western dietary pattern [32]. Because Cu-rich foods such as meats, nuts, seeds, and cereals are commonly consumed in Iranian traditional dietary pattern, therefore, relatively the lower proportion of inadequate Cu intake in the studied subjected could be explained [9]. However, the interaction between dietary Cu and fructose is suggested to be considered for the possible synergistic effects on NAFLD pathology [8].

Our results showed that serum liver enzymes and ferritin were significantly higher in patients with NAFLD compared with the controls (Table 2). However, we failed to present any significant differences in serum Cu and ceruloplasmin concentrations between the groups as well as any association between Cu and ceruloplasmin status and NAFLD (p = 0.443 and p = 1.000, respectively). Moreover, serum Cu deficiency (< 70 µg/dl) was found in 6.3% of controls and 2.5% of cases while only 1.3% of each group showed low serum ceruloplasmin concentration (> 20 g/L). A number of animal and human studies have indicated low serum levels of Cu in association with not only fat accumulation in the liver [11–13], hypertriglyceridemia and cholesterolemia [33], but also a number of chronic diseases [20]. For example, a significant relationship was observed between higher Cu concentration and lower risk of NAFLD in men in China (P for trend < 0.001) [34]. Serum ceruloplasmin might even help in NAFLD diagnosis among children [17]. However, a study on 95 patients with NAFLD revealed a significant association between low serum ceruloplasmin < 25 mg/dl) and lower BMI (p = 0.005) [13]. Moreover, higher Cu concentrations have been shown in cirrhosis and hepatocarcinoma [18]. Therefore, it appears that both deficiency and excessive serum levels of Cu appears to be linked with steatosis in the liver. Recent evidence indicates the association between marginal Cu and NAFLD and/or obesity as mild Cu deficiency was reported to be linked with hypercholesterolemia in humans in 1984 [27, 35–37]. Findings from a number of studies demonstrating the relationship between Cu deficiency and dyslipidemia have been explained through the low intestinal absorption of Cu in NAFLD and metabolic syndrome [12, 34, 38, 39].

We recently have reported serum Cu and ceruloplasmin were significantly and positively correlated with serum TG and LDL-C levels which was in accordance with a rat model study on the association between mild Cu deficiency and serum TC levels [40, 41].

These findings have suggested following Cu deficiency, not only increased cholesterol synthesis but also reduced cholesterol degradation lead to dyslipidemia [42, 43]. Regulatory factors of fatty acid (FA) and cholesterol such as sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) are modulated by Cu deficiency [44, 45]. Furthermore, this condition could be partly explained through acyl CoA pathway which is down-regulated in the intestine leading to reduced mitochondrial and peroxisomal beta-oxidation and availability of FAs as well as increased cytoplasmic FA [8]. Nevertheless, further investigation is required to understand the underlying mechanism.

In the present study, serum and dietary Cu were correlated with obesity indices whereas serum ceruloplasmin was significantly related with WC and ferritin level only in patients with NAFLD. To define predictors of NAFLD, results of multivariate logistic regression revealed that after controlling the confounders including BMI, WC, serum levels of ALT, AST and ferritin, low Cu intake (< 0.95 mg/day) was more likely to increase the odds of NAFLD (OR = 5.82, CI 95%: 1.55, 21.85, p = 0.009) (p for trend = 0.002). This finding agreed with the previous evidence illustrating that increased BMI, ALT and AST) are associated NAFLD risks [2, 46]. Nevertheless, studies investigating the role of Cu in in NAFLD are limited. Spahis et al. [47] showed that Cu status (both deficiency and increase) is associated with some conditions including lipid peroxidation, inflammation, and oxidative stress which these disturbances are common in NAFLD. Indeed, reduced Cu appears to play a role in NAFLD through the link with dietary fructose and NAFLD complex nature shown in both animal [14, 31] and human experiments [13, 33, 34, 48]. We should recognize that Cu deficiency was not common, particularly in cases, in our studied population, probably because of relatively varied diet they consumed.

Limitations of the study

In the present study, we tried to examine the Cu-NAFLD link in humans through designing a case-control study as well as assessing dietary Cu status parallel to Cu biomarkers to find the predictors for NAFLD, particularly by considering possible confounders, nevertheless, future human studies are needed to justify Cu status in relation to key hits in the pathophysiology of NAFLD by assessing the interaction between Cu and other minerals.

Conclusion

It is concluded that Cu inadequacy could be considered as a NAFLD predictor. More mechanistic studies are necessary to show the role of Cu in NAFLD.

Abbreviations

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BMI

Body mass index

CI

Confidence interval

Cu

Copper

FBS

Fasting blood sugar

FFAs

Free fatty acids

HC

Hip circumference

HDL-C

High-density lipoprotein cholesterol

IFCC

International federation of clinical chemistry and laboratory medicine

IR

Insulin resistance

LDL-C

Low-density lipoprotein cholesterol

NAFLD

Non-alcoholic fatty liver disease

NASH

Non-alcoholic steatohepatitis

ROS

Reactive oxygen species

SOD

Super oxide dismutase

T2DM

Type 2 diabetes mellitus

TC

Total cholesterol

Tf

Transferrin

TG

Triglyceride

WC

Waist circumference

WHO

World health organization

WHR

Waist to hip ratio

WHtR

Waist to height ratio

Author contributions

The authors’ responsibilities were as follows: S.A. and S.Z.G. collected data and wrote the original paper; M.E.M. S.R.A. and H.T. contributed to the conception of the article; M.E.M. and S.A.contributed to the statistical analysis; H.T. and M.E.M. contributed to the final revision of the manuscript. All authors read and approved the final version of the manuscript.

Funding

This study was funded by the ‘Research Vice-Chancellor’ of Tabriz University of Medical Sciences, Tabriz, Iran (Grant no.64611).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All procedures performed in this study were in accordance with the ethical standards of the Ethics Committee of Tabriz University of Medical Science. The study protocol obtained approval from the Ethics Committee of Tabriz University of Medical Science (IR.TBZMED.REC.1399.176). Informed written consent was obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.